Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow

To improve ultrasound contrast agents targeted to the adhesion molecules P-selectin and VCAM-1 for the purpose of molecular imaging of atherosclerotic plaques, perfluorocarbon-filled phospholipid microbubble contrast agents were coupled by a polyethylene glycol–biotin–streptavidin bridge with mAb MVCAM.A(429), a sialyl Lewisx polymer (PAA-sLex), or both (dual). Approximately three hundred thousand antibody molecules were coupled to the surface of each microbubble. Recombinant mouse P-selectin and/or VCAM-1 coated on flow chambers showed saturation of binding at approximately 15 ng/μl, resulting in 800 and 1200 molecules/µm2 for P-selectin and VCAM-1, respectively. Dual substrates coated with equal concentrations of P-selectin and VCAM-1 had site densities between 50 and 60% of single substrates. When microbubbles were perfused through flow chambers at 5 × 106 microbubbles/ml (wall shear stress from 1.5 to 6 dyn/cm2) dual-targeted microbubbles adhered almost twice as efficiently as single-targeted microbubbles at 6 dyn/cm2. The present study suggests that dual-targeted contrast agents may be useful for atherosclerotic plaque detection at physiologically relevant shear stresses.

Graphical abstractMicrobubble adhesion efficiency within a parallel plate flow chamber. Attachment efficiency (adherent microbubbles/all microbubbles passing field of view per 45 s) of dual, MVCAM.A(429)-targeted microbubbles and PAA-sLex microbubbles as a function of shear stress. “*”, p < 0.05 from sLex microbubbles. “+”, p < 0.05 from MVCAM.A(429) microbubbles. Error bars, SEM for n = 8.Figure optionsDownload full-size imageDownload as PowerPoint slide